Method fo assessing antidepressant drug therapy via transport inhibition of monoamine neurotransmitters

ABSTRACT

A method of measuring the individual response to antidepressant drug therapy on the transport inhibition of monoamine neurotransmitters involves in vitro monitoring of radiolabeled monoamine neurotransmitter transport into cells transfected with transport proteins similar to those on neural cells of the individual being studied. The transport occurs in unbuffered serum of the individual who is undergoing or will later undergo pharmaceutical treatment for depression or other neuropsychiatric disorders. The use of buffers is avoided so that the sensitive balance of bound/free drug within the individuals serum is not disrupted prior to or during testing.

FIELD OF THE INVENTION

[0001] The invention relates to a method of measuring the transport ofmonoamine neurotransmitters, and further of using the method todetermine efficacious dosages of medications for treatment ofneuropsychiatric disorders in humans.

BACKGROUND OF THE INVENTION

[0002] Neurotransmitters are small molecules that carry informationacross a synapse from the end of one nerve cell to the end of anothernerve cell. The neurotransmitters are a diverse group of chemicalcompounds ranging from simple amines to amino acids to polypeptides. Themechanisms by which they elicit responses in both presynaptic andpostsynaptic neurons are similarly diverse. Much research has beenconducted on monoamine neurotransmitters such as serotonin,norepinephirine, and dopamine. These neurotransmitters are believed tohave important effects on mood and behavior.

[0003] Dysregulations in monoamine neurotransmitter systems arecurrently believed to play a seminal role in the neurobiologicalunderpinnings of certain neuropsychiatric disorders, includingdepression, anxiety, schizophrenia, eating disorders, Parkinson'sdisease and sleeping disorders. The actions of these monoamineneurotransmitters are typically terminated by transport (also know asreuptake) of the transmitter back into the cell of origin via cellsurface proteins known as transporters.

[0004] Treatment of neuropsychiatric disorders often involvesmanipulation of neurotransmitter transport systems with pharmaceuticalagents that inhibit or antagonize a particular transporter. Applicationof an appropriate antagonist to block uptake prolongs and enhances theaction of the neurotransmitter. For example, the serotonin andnorepinephrine reuptake blockers, paroxetine and desipramine,respectively, are used as antidepressants.

[0005] Determination of the amount of transporter antagonism as afunction of drug dose or blood concentration is most successfullystudied using in vivo techniques such as positron emission tomography(PET). However, PET is a very invasive procedure that utilizesradioactive isotopes injected into the body and requires access tonearby state of the art research facilities. Because it is undesirableand difficult to use in vivo studies, the effectiveness of newmedications or of particular doses of known medications are typicallydetermined empirically by treating human patients and trying to observediscrete measures of therapeutic effectiveness as a function of drugdosage.

[0006] The potency of individual drugs to antagonize monoaminetransporters can be determined in vitro using cultured cells. In suchtests, cells are cultured that contain the transporters corresponding tothe neurotransmitters under study. The cells are stabilized in a buffersolution appropriate for maintaining the vitality of the cells. Aradioactive form of the neurotransmitter and the proposed inhibitor areadded to the buffer, and transport of the radiolabeled neurotransmitteris monitored. Transport with and without the presence of variousconcentrations of the proposed inhibitor are compared and an overallpercentage of inhibition is determined.

[0007] Traditional in vitro methods of studying neurotransmittertransport have heretofore provided general insight into theunderstanding of how antidepressants inhibit neurotransmittertransporters and how potent a given agent may be, but the traditionalstudies are often inaccurate and based upon vague correlations betweenoverall serum drug concentrations and the predicted transport blockageprovided by the drugs in the body.

[0008] Individual variations in response to treatment with transportinhibiting antidepressant drugs is little studied and not wellunderstood. Often, to compensate for individual responses, drugs are, asnoted above, simply administered to an individual in varyingcombinations and dosages until the most significant reduction insymptoms are observed. Such methods of determining optimum dosages areuncertain, take long periods of time, and often result in a patientbeing over or under-medicated for extended lengths of time.

[0009] It is desired to provide an in vitro method of studying theeffectiveness of monoamine transporter inhibitors that is patientspecific and that provides data useful in determining the most effectivedose of inhibitor or combination of inhibitors for treatment of aneurological disorder. Such a technique should be minimally invasive andshould provide an objective guidance in the determination of optimumdrug combinations and dosages.

BRIEF SUMMARY OF THE INVENTION

[0010] The invention is a method of determining the effectiveness oftransport inhibiting antidepressant medication upon neurotransmittertransport proteins and the uptake of monoamine neurotransmitters bycells. The method is particularly effective for determining theeffectiveness of drug treatment on an individual basis in view of thevarying efficacy of antidepressant drugs among individuals within thesame species. The method is effective for testing an individual subjectundergoing drug treatment for a neuropsychiatric disorder, who is beingtreated with at least one suspected monoamine neurotransmitter transportinhibitor.

[0011] The inventors have found that buffers tend to significantly alterthe equilibrium of protein-bound drug to free drug within the serum, andvariation of the bound/free drug ratio negatively affects the accuracyof the test method. Therefore, in accordance with the invention, nobuffer solutions are added to the serum prior to or during testing,except for de minimis amounts of buffer required as a carrier forintroduction of radiolabeled neurotransmitters, as discussed below.

[0012] To implement the method, a test subject is first chosen. A cellculture is developed that has transport characteristics similar to acell system of interest. Most typically, the cell system of interestcomprises the neural cells of the subject.

[0013] A baseline serum sample is derived from blood drawn from thesubject prior to administration of the drug under study. After thebaseline serum sample is obtained, a controlled dosage of drug fortreatment of depression or another neurological disease is administeredto the subject. Serum is derived from blood samples drawn from thesubject after administration of the drug. The drug-containing serumcontains the amount of drug normally present within the serum of thetreated subject.

[0014] The cells in culture are divided into two test cultures. Thefirst culture, labeled the baseline culture, is used to further culturethe baseline serum from the subject. The second culture, labeled thedrug-containing culture, is used to further culture the actualdrug-containing serum drawn from the subject. After allowing therespective cells and serum samples to come in contact with each otherfor a short period of time, a known amount of labeled monoamineneurotransmitter is added to the serum of each culture. The amount ofcellular uptake of the labeled neurotransmitter is then measured foreach culture.

[0015] The overall effectiveness of the inhibitory drug may bedetermined by comparing the measured uptake of labeled neurotransmitterin the baseline culture compared to that of the drug-containing culture.The percentage of transporter occupancy is calculated as the fraction ofmeasured neurotransmitter uptake in the drug-containing sample dividedby the measured neurotransmitter uptake in the baseline sample,multiplied by 100.

[0016] The invented method recognizes that transport inhibition isdependent upon the amount of free drug available for transportinhibition within the serum of any particular individual. The methodalso recognizes that use of buffered solutions will disturb the amountof free drug available in the serum. This property has not beenrecognized by previous methods of measuring transport inhibition.

[0017] Transport testing in the past has emphasized the use ofbuffer-washed platelet preparations, buffered serum solutions, orbuffered non-serum solutions as preparation solutions or mediums forcell cultures. Heretofore, actual transport studies have been conductedin complex, buffered salt solutions. If serum has been used as a medium,buffers have always been added to the serum medium in an effort topreserve the medium.

[0018] Use of buffers to manipulate or handle drug-containing serum,before or during in vitro transport of neurotransmitters, disrupts thenatural equilibrium of protein-bound drug and free drug within the serumsample, thus distorting results of any study based upon such anexperiment. By using serum of the tested individual to be tested,without the addition of buffers, as the medium for in vitro transporttrials, the natural balance of protein-bound drug and free drug ismaintained and an accurate uptake profile for that individual may bedetermined.

[0019] The method of analyzing drug efficacy (amount of transporterblockade) using the unbuffered serum as a test medium has broad rangingapplication. For instance, effective individual treatment regimens maybe designed for individual patients suffering depression. Such regimensare designed by objectively determining the effect of transportinhibition under varying drug treatments. In the past, individualresponses to drug treatment were measured by measures of illnessseverity such as the Montgomery-Asberg Depression Rating Scale or theHamilton Depression Rating Scale. The invented method allows much moredirect and accurate in vitro determinations of antidepressant drugefficacy in individuals.

[0020] By way of example, according to one embodiment of the inventedmethod, a human patient having symptoms of depression might have abaseline serum sample prepared and then be treated with a standarddosage of paroxetine, a serotonin transporter inhibitor. If theparoxetine is ineffective in alleviating the depression of the patient,a serum sample is obtained and transport inhibition is tested using thedescribed method. If the assay reveals that the drug is providingsignificant serotonin transport inhibition (typically about 80% SERTblockage), then the treatment of this individual's depression may bedeemed to be not responsive to blockade of serotonin uptake, and anyunnecessary dosage increases of serotonin uptake inhibitor may beavoided. If the assay reveals that the drug is providing insufficientserotonin transport inhibition, then the source of continued depressionmay be the result of insufficient dosage and/or concentration of freedrug available within that individual, so an increased dosage of thedrug may be indicated. Moreover, this method can determine the amount oftransport blockade at other transporters as well. In the example above,a patient not responding to serotonin transport inhibition may benefitby addition of a drug that also blocks norepinephrine transport.

[0021] Guidance provided by this assay avoids much of the unnecessaryguesswork previously associated with determining proper medicationlevels for individuals suffering neurological disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Having thus described the invention in general terms, referencewill now be made to the accompanying figures, which are not necessarilydrawn to scale, and wherein:

[0023]FIG. 1 is a graph showing the relationship between serotoninuptake and concentrations of serum paroxetine as measured in accordancewith an embodiment of this invention;

[0024]FIG. 2 is a graph showing the relationship between serotoninuptake and concentrations of serum paroxetine as measured by PET imagingof a human brain; and

[0025]FIG. 3 is a graph showing the relationship between norepinephrineand serotonin uptake and concentrations of paroxetine and desipramine inindividual unbuffered human serum samples of patients receivingmedication for major depressive disorder in accordance with anembodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The invention will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, the invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements.

[0027] To determine the effective monoamine neurotransmitter reuptakeinhibition (transporter occupancy) of a particular subject undertreatment with a particular drug dosage, a baseline measurement isdetermined in accordance with the techniques described below based uponserum derived from drug-free blood drawn from the subject, anddrug-containing measurement is determined in accordance with thetechniques described below based upon serum derived from blood drawnfrom the subject when the subject is undergoing drug therapy with acontrolled dosage of one or more suspected antidepressant compositions.The serum samples may be freshly drawn or may be frozen and stored forsubsequent testing.

[0028] For each serum sample, the serum is used as a medium forexamining the transport of neurotransmitters into human transportertransfected cells. Labeled neurotransmitters are added to the serummedium and uptake of the neurotransmitters is measured. The amount ofuptake varies with the amount of free drug present within the serum. Thecalculated transport inhibition (transporter occupancy) is determined asthe quotient of the measured neurotransmitter uptake of thedrug-containing sample divided by the measured neurotransmitter uptakeof the drug-free sample (then multiplied by 100 to convert the fractionto a percentage).

[0029] The baseline sample is preferably derived from the subjects' owndrug-free blood. However, if such a sample is not available, the testmay be run using a drug-free pooled serum sample in place of thebaseline sample. The drug-free pooled serum sample should haveapproximately the same transport inhibitory function as the drug-freeserum sample of a subject.

[0030] For study of monoamine neurotransmitter transport, cell culturesare transfected with transporters selected from serotonin transporter(SERT), norepinephrine transporter (NET), or dopamine transporter (DAT).A cell culture producing a stable expression of the human SERT, NETand/or DAT, and having high-affinity, Na⁺-dependent transport ofserotonin, norepinephrine, or dopamine with pharmacological propertiesidentical with those of native membranes can be used.

[0031] The transfected cell cultures are incubated, separately, with thebaseline and drug-containing serum test media. After incubation, small,controlled amounts of radiolabeled monoamine neurotransmitters are addedto the cultures. Small amounts of buffer are required at this stage inorder to carry the radiolabeled neurotransmitters and to introduce theradiolabeled neurotransmitters to the test media.

[0032] The neurotransmitters, which are preferably [³H]-labeledvariations of serotonin, norepinephrine, or dopamine, are allowed timeto be transported into the cells. The cells are then washed of excessradioactivity and overall uptake of labeled neurotransmitter ismeasured, and the measurements for the samples are compared to determinethe transport inhibition in the drug-containing sample

[0033] Little or no buffers should be added to the serum from the timethe serum is drawn until the time when the cells are washed followinguptake of the neurotransmitters from the serum medium, except the smallamount of buffer used as a carrier for the radiolabeledneurotransmitters mentioned above. Stabilizing buffers or preservativesdisrupt the ratio of free drug to protein-bound drug present in theserum, thus introducing flaws in the analysis of drug activity. As usedherein, the term “buffer-free” and similar terms indicate that buffer isabsent from the solution or present in such quantities that the bufferhas no measurable effect on drug binding to proteins within thesolution. As a practical matter, buffers preferably constitute less thanabout 2% by volume of the assay medium. Use of such small amounts of thebuffers have no appreciable effect upon the measured transportinhibition.

[0034] This method is effective in the study of various monoamineneurotransmitters, including but not limited to serotonin,norepinephrine, and dopamine. The method is applicable to pharmaceuticalcompositions that inhibit the transport of monoamine neurotransmittersacross cell membranes, particularly noradrenergic reuptake inhibitors,5-HT reuptake inhibitors, dopamine reuptake inhibitors, and/orcombinations thereof.

[0035] A comparison of neurotransmitter uptake in the drug treated serumwith a base line sample of drug-free serum drawn from the subjectprovides a measurement of percent inhibition due to presence of thedrug. Based upon such an outcome, overall effectiveness of the drugtreatment may be examined and compared to outcomes with other drugdosages or combinations. Drug dosage or choice of drug may then beadjusted accordingly.

[0036] Whereas test conditions typically vary between test runs, forexample variations in the cell culture, serum preparation, and othertest conditions, it is preferred that each serum sample be tested inparallel with the baseline sample, so that the two may be compared and apercent inhibition determined. It is possible, and within the scope ofthis invention, however, that the test methods could be finely tuned andcalibrated in order to determine a percent inhibition of adrug-containing serum sample without the need to compare the testedsample with a baseline sample.

[0037] Because unbuffered human serum is the vehicle in the assay, thefree transporter-inhibiting drug in the serum samples would be expectedto reflect the drug concentration in the patients' cerebrospinal fluid(CSF). In order to demonstrate the accuracy of correlation between thetransporter occupancy predicted by the invented test method and actualtransporter occupancy occurring in the brain of a treated subject,occupancy predicted by this method was compared to actual occupancyobserved using PET imaging.

[0038] Referring to FIG. 1, pooled buffer-free human serum was spikedand equilibrated, for 2 hours at 37° C. with continuous mixing, withparoxetine. 0.3 ml of the serum was directly used for the 5-HT uptakeassay in accordance with the invention. The test was repeated forseveral varying concentrations of paroxetine. FIG. 1 shows a graph ofparoxetine concentration versus the percentage of uptake inhibition(transporter occupancy) predicted by the invented method.

[0039] Referring to FIG. 2, a graph showing serum paroxetineconcentration versus actual human brain transporter occupancy wasobtained from Meyer et al., 2001. American Journal of Psychiatry158:1843-1849. The graph shows the results measured by PET imaging usingPET ligand ^([11)C^(])DASB.

[0040] Comparing FIG. 1 with FIG. 2, it has been demonstrated that thetransporter occupancy actually occurring within the brain (FIG. 2) isaccurately reflected by the invented method (FIG. 1). Although theaccuracy is demonstrated for paroxetine, it is reasonable to concludethat the transport inhibition produced by other reuptake inhibitingdrugs are accurately reflected by the invented method as well.

EXAMPLES

[0041] In order to demonstrate the assay method of the invention, themethod was used to determine the uptake of serotonin and norepinephrinein the presence of antidepressant drugs peroxetine and desipramine.

[0042] Background of Experiment

[0043] In an open-label, parallel-group, forced-titration study, 52outpatients with DSM-IV major depressive disorder and a baselineMontgomery Asberg Depression Rating Scale score >20 were randomlyassigned to treatment with paroxetine (to 60 mg/day) or desipramine (to300 mg/day) in a 3-to-1 ratio, respectively. Norepinephrine and 5-HT(serotonin) transporter function were assayed by using human transportertransfected cells in the presence of serum collected at baseline and theend of each treatment week. Data from 36 patients were analyzed.

[0044] The tests were conducted in a modified monoamine uptake assay bymeasuring uptake inhibition of norepinephrine and 5-HT in serum samplestaken from patients treated with escalating doses of paroxetine ordesipramine. This method maintained the important equilibrium betweenfree and serum-protein-bound drug, hence modeling in vivo conditionswhere only free drug is accessible to the brain and clinically relevantsites of action.

[0045] This was a multicenter, open-label, parallel-group,forced-titration study of paroxetine or desipramine in patients with adiagnosis of major depressive disorder. Patients were recruited byphysicians at six outpatient centers. Medication assignment wasdetermined by means of a treatment assignment sheet provided to eachcenter such that for every four patients assigned to study treatment,three were assigned to receive paroxetine and one was assigned toreceive desipramine. The 7-week dosing schedule for paroxetine was 10mg/day for 1 week, 20 mg/day for 2 weeks, 40 mg/day for 2 weeks, and 60mg/day for 2 weeks. The 7-week dosing schedule for desipramine was 50mg/day for 1 week, 100 mg/day for 2 weeks, 200 mg/day for 2 weeks, and300 mg/day for 2 weeks.

[0046] Safety and efficacy assessments were made in accordance withspecifications generally accepted and approved for testing ofpharmaceutical effect on neurological disorders, as further specified inAm J Psychiatry 2002; 159:1702-1710.

[0047] Analysis of Serotonin Uptake in Human Serum dosed with Paroxetine

[0048] A cell culture of HEK-293 (human embryonic kidney) cells having astable transfection of human SERT (serotonin transporter) cDNA, andhaving high-affinity, Na+-dependent transport of serotonin withpharmacological properties identical with those of native membranes wasprovided by Dr. Randy Blakely of Vanderbilt University.

[0049] A first volume of baseline serum was derived from blood takenfrom the individuals at the beginning of the study. The drug-free serumwas frozen and portions of the serum were thereafter unfrozen and usedas needed. In each experiment, the baseline samples and allconcentrations of paroxetine were assayed in triplicate in a single24-well plate for each cell line.

[0050] Transporter uptake was assayed by aspirating the cell culturemedia and washing the plated cells with 0.5 ml of phosphate-bufferedsaline (pH=7.2) (Sigma, St. Louis, Mo.). Three hundred microliters ofeach serum sample was loaded into triplicate wells and preincubated forexactly 5 minutes at 37° C., after which 10 μl of [³H]5-HT (40 nM finalconcentration; Amersham, Piscataway, N.J.) was added and the sample wasincubated at 37° C. for an additional 5 minutes. The assay wasterminated by aspirating the serum and washing the cells with 1.0 ml ofphosphate-buffered saline. The cells were lysed with 500 μl of 0.1 Msodium hydroxide, and 450 μl was transferred to liquid scintillationvials. The [³H]-5-HT uptake of the cells was quantified on ascintillation counter at 50% efficiency.

[0051] The measured [³H]-5-HT uptake of each drug-containing sample wasthen compared to its corresponding baseline sample and the amount ofuptake inhibition was calculated. The results are plotted in the lowerleft quadrant of FIG. 3.

[0052] Analysis of Serotonin Uptake in Human Serum dosed withDesipramine

[0053] A cell culture of HEK-293 (human embryonic kidney) cells having astable transfection of human SERT (serotonin transporter) cDNA, andhaving high-affinity, Na+-dependent transport of serotonin withpharmacological properties identical with those of native membranes wasprovided by Dr. Randy Blakely of Vanderbilt University.

[0054] A first volume of baseline serum was derived from blood takenfrom the individuals at the beginning of the study. The drug-free serumwas frozen and portions of the serum were thereafter unfrozen and usedas needed. In each experiment, the baseline samples and allconcentrations of desipramine were assayed in triplicate in a single24-well plate for each cell line.

[0055] Transporter uptake was assayed by aspirating the cell culturemedia and washing the plated cells with 0.5 ml of phosphate-bufferedsaline (pH=7.2) (Sigma, St. Louis, Mo.). Three hundred microliters ofeach serum sample was loaded into triplicate wells and preincubated forexactly 5 minutes at 37° C., after which 10 μl of [³H]5-HT (40 nM finalconcentration; Amersham, Piscataway, N.J.) was added and the sample wasincubated at 37° C. for an additional 5 minutes. The assay wasterminated by aspirating the serum and washing the cells with 1.0 ml ofphosphate-buffered saline. The cells were lysed with 500 μl of 0.1 Msodium hydroxide, and 450 μl was transferred to liquid scintillationvials. The [³H]-5-HT uptake of the cells was quantified on ascintillation counter at 50% efficiency.

[0056] The measured [³H]-5-HT uptake of each drug-containing sample wasthen compared to its corresponding baseline sample and the amount ofuptake inhibition was calculated. The results are plotted in the lowerright quadrant of FIG. 3.

[0057] Analysis of Norepinephrine Uptake in Human Serum dosed withParoxetine

[0058] A cell culture of HEK-293 (human embryonic kidney) cells having astable transfection of human NET (norepinephrine transporter) cDNA, andhaving high-affinity, Na+-dependent transport of norepinephrine withpharmacological properties identical with those of native membranes wasprovided by Dr. Randy Blakely of Vanderbilt University.

[0059] A first volume of baseline serum was derived from blood takenfrom the individuals at the beginning of the study. The drug-free serumwas frozen and portions of the serum were thereafter unfrozen and usedas needed. In each experiment, the baseline samples and allconcentrations of paroxetine were assayed in triplicate in a single24-well plate for each cell line.

[0060] Transporter uptake was assayed by aspirating the cell culturemedia and washing the plated cells with 0.5 ml of phosphate-bufferedsaline (pH=7.2) (Sigma, St. Louis, Mo.). Three hundred microliters ofeach serum sample was loaded into triplicate wells and preincubated forexactly 5 minutes at 37° C., after which 10 μl of [³H]norepinephrine (40nM final concentration; Amersham, Piscataway, N.J.) were added and thesample was incubated at 37° C. for an additional 5 minutes. The assaywas terminated by aspirating the serum and washing the cells with 1.0 mlof phosphate-buffered saline. The cells were lysed with 500 μl of 0.1 Msodium hydroxide, and 450 μl was transferred to liquid scintillationvials. The [³H]-norepinephrine uptake of the cells was quantified on ascintillation counter at 50% efficiency.

[0061] The measured [³H]-NE uptake of each drug-containing sample wasthen compared to its corresponding baseline sample and the amount ofuptake inhibition was calculated. The results are plotted in the upperleft quadrant of FIG. 3.

[0062] Analysis of Norepinephrine Uptake in Human Serum dosed withDesipramine

[0063] A cell culture of HEK-293 (human embryonic kidney) cells having astable transfection of human NET (norepinephrine transporter) cDNA, andhaving high-affinity, Na+-dependent transport of norepinephrine withpharmacological properties identical with those of native membranes wasprovided by Dr. Randy Blakely of Vanderbilt University.

[0064] A first volume of baseline serum was derived from blood takenfrom the individuals at the beginning of the study. The drug-free serumwas frozen and portions of the serum were thereafter unfrozen and usedas needed. In each experiment, the baseline samples and allconcentrations of desipramine were assayed in triplicate in a single24-well plate for each cell line.

[0065] Transporter uptake was assayed by aspirating the cell culturemedia and washing the plated cells with 0.5 ml of phosphate-bufferedsaline (pH=7.2) (Sigma, St. Louis, Mo.). Three hundred microliters ofeach serum sample was loaded into triplicate wells and preincubated forexactly 5 minutes at 37° C., after which 10 μl of [³H]norepinephrine (40nM final concentration; Amersham, Piscataway, N.J.) were added and thesample was incubated at 37° C. for an additional 5 minutes. The assaywas terminated by aspirating the serum and washing the cells with 1.0 mlof phosphate-buffered saline. The cells were lysed with 500 μl of 0.1 Msodium hydroxide, and 450 μl was transferred to liquid scintillationvials. The [³H]-norepinephrine uptake of the cells was quantified on ascintillation counter at 50% efficiency.

[0066] The measured [³H]-NE uptake of each drug-containing sample wasthen compared to its corresponding baseline sample and the amount ofuptake inhibition was calculated. The results are plotted in the upperleft quadrant of FIG. 3.

[0067] Many modifications and other embodiments of the inventions setforth herein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

That which is claimed:
 1. An assay method for identifying the efficacyof antidepressant drugs which inhibit the cellular uptake of monoamineneurotransmitters, comprising obtaining a buffer-free serum sample froman individual undergoing treatment with one or more antidepressant drug;obtaining a cell culture having neurotransmitter transportcharacteristics similar to the neurotransmitter transport characteristicwithin neural cells of said individual; maintaining the cell culturewithin the buffer-free drug-containing serum; introducing a labeled doseof monoamine neurotransmitter to the drug-containing serum; andmeasuring the uptake of labeled neurotransmitter into cells of theculture.
 2. The assay method of claim 1, further comprising the step ofcomparing the uptake of the labeled neurotransmitter with a baselineuptake value for the individual in the absence of antidepressant drug,thereby determining the percentage of neurotransmitter inhibition due tothe drug dosage present within the drug-containing serum sample.
 3. Theassay method of claim 1, further comprising the step of repeating themethod for serum samples containing varying dosages of antidepressantdrug in order to determine the most efficacious dose for the individual.4. The assay method of claim 1, wherein the one or more antidepressantdrug is one or more compositions that inhibit the transport of monoamineneurotransmitters.
 5. The assay method of claim 1, wherein the monoamineneurotransmitters are selected from serotonin, norepinephrine, dopamine,and combinations thereof.
 6. The assay method of claim 1, wherein theantidepressant drugs are selected from the group consisting ofnoradrenergic reuptake inhibitors, 5-HT reuptake inhibitors, dopaminereuptake inhibitors, and combinations thereof.
 7. The assay method ofclaim 1, wherein no more than 2% (v/v) of buffer is introduced to thedrug-containing serum at any time during the assay.
 8. An assay methodfor identifying the efficacy of antidepressant drugs which inhibit thecellular uptake of monoamine neurotransmitters, comprising obtaining abuffer-free serum sample from an individual undergoing treatment withone or more monoamine neurotransmitter reuptake inhibitor; obtaining acell culture having neurotransmitter transport characteristics similarto the neurotransmitter transport characteristic within neural cells ofsaid individual; maintaining the cell culture within the buffer-freeinhibitor-containing serum; introducing a labeled dose of monoamineneurotransmitter to the drug-containing serum; and monitoring the uptakeof labeled neurotransmitter into cells of the culture.
 9. The assaymethod of claim 8, wherein the monoamine neurotransmitter reuptakeinhibitor is selected from the group consisting of paroxetine anddesipramine.
 10. An assay method for identifying the efficacy ofantidepressant drugs which inhibit the cellular uptake of monoamineneurotransmitters, comprising the step of measuring the uptake oflabeled monoamine neurotransmitter into cells of a culture whilemaintaining the culture in a buffer-free serum sample.
 11. The assaymethod of claim 10, wherein the cells of the culture haveneurotransmitter transport characteristics similar to theneurotransmitter transport characteristic within neural cells of ahuman.